Engineering Probiotic E. coli Nissle 1917 for Release of Therapeutic Nanobodies

Bioengineered probiotics enable new opportunities to improve cancer treatment strategies due to their tumor-colonizing capabilities. Here, we will describe the development of a probiotic E. coli Nissle 1917 platform encoding a synchronized lysis mechanism for the localized and sustained release of blocking nanobodies against immune checkpoint molecules like programmed cell death protein-ligand 1 and cytotoxic T lymphocyte–associated protein-4. Specifically, we will detail the experimental protocols needed to (1) encode and validate binding of recombinantly produced checkpoint blockade nanobodies, (2) evaluate the therapeutic efficacy and safety of the probiotic platform in syngeneic tumor-bearing mice, and (3) analyze the immunophenotype of the tumor microenvironment.